Abstract: Single radial hemolysis test for the detection of antibodies against Boophilus microplus was developed. Optimum test conditions were determined on the basis of clear hemolysis and maximum haemolytic zone diameter by using human, sheep and rabbit erythrocytes; complement dilutions and temperature. There was a non significant difference (P>0.05) in average haemolytic zone diameters among the erythrocytes of three species used but qualitative analysis indicated that human group "O" erythrocytes at the rate of 2 per cent in agar gel medium gave better results. A 1:2 dilution of complement in vernal buffer at the rate of 8 percent in agar gel medium was found to be optimally appropriate. The combination of over night refrigeration and subsequent incubation at 37 °C in a humid chamber for 8-12 hours gave good results regarding the quality of the zones of hemolysis which were well delineated.